Apparatus and method for maintaining and growth biological cells

ABSTRACT

An apparatus and related method are disclosed, for receiving, maintaining and growing biological cells ex vivo within a portable cassette, without exposing the cells to the external environment. The portable cassette is used in combination with a processor instrument that facilitates an initial inoculation of the cassette with cells of the kind to be grown and subsequently distributes those cells in a predetermined pattern (e.g., uniformly) throughout a cell growth chamber. Thereafter, the portable cassette is used in combination with an incubator instrument that incubates the cell growth chamber so that the cells are optimally expanded. The same processor instrument then is used to harvest the expanded cells from the portable cassette. Both instruments are configured to condition the portable cassette during stages of the cell growth process, without disturbing the cassette&#39;s sterile system. In addition, an updatable memory device associated with the cassette stores significant information about the cassette and its condition during the various steps of the cell growth process. Such information is useful both for subsequent archival purposes and for facilitating a resumption of the cell growth process in the event of any instrument failure or significant alarm condition.

This application is a continuation, of application Ser. No. 08/483,517filed Jun. 7, 1995, now abandoned.

BACKGROUND OF THE INVENTION

1. Introduction

This invention relates generally to apparatus for maintaining andgrowing biological cells ex vivo and, more particularly, to apparatus ofthis kind that maintains and grows the cells in a portable cassettewhile maintaining a sterile system that is closed to the externalenvironment.

Many medical disorders can now be resolved by using transplanted cells,tissues, or organs. Transplantation has evolved from the surgicaltransfer of tissue from one part of a patient's body to another, to thesurgical transfer of organs and tissues between individuals, to thetransplantation of blood and immune systems between individuals. Withincreased demonstration of the medical benefit of tissuetransplantation, the demand for organs and tissues suitable for theseprocedures has far exceeded the availability. Furthermore, in thosecases where availability is less an issue, e.g., bone marrow, theprocedure is cost prohibitive and can be invasive for the donor or thepatient.

As an evolution of the clinical need, two related fields have evolved,which have been termed “cell therapy” and “tissue engineering.” Celltherapy generally refers to the use of living cells, rather than drugs,to treat a clinical disorder or disease. Perhaps the most widelypracticed form of cell therapy today is with bone marrow orhematopoietic stem cell transplantation in patients who have receivedhematopoietic toxic chemotherapy or radiation. This procedure involvesthe reinfusion of early stage cells that originate in the bone marrow,so that these cells can reestablish a patient's blood and immune system,and often the bone marrow tissue as well. Through this cell therapyprocess, the hematopoietic toxicity from cancer treatment is remedied.

Tissue engineering generally refers to the utilization of differentdisciplines between engineering, physiology, and cell biology, todevelop at least a partially living tissue that is capable of normaltissue function. Once produced, this tissue may be transplanted intohumans to restore or improve normal tissue or organ function. Numerousbiotechnology companies are engaged in projects to engineer humantissues for transplantation.

For both cell therapy and tissue engineering procedures, there is acritical need to be able to process and/or produce ex vivo the cellsthat will be used for the therapeutic transplant. Biological science hasnow progressed such that, for many of the human tissues, methodology hasbeen developed so that the key cells of that tissue can be grown outsidethe body. As a result, a clinically useful amount of tissue can begenerated from a small amount of starting material, which is obtainedwith a minimally invasive technique. With this achievement, theopportunity for increasing and more diverse use of tissuetransplantation is offered.

In parallel with this advancement, and largely dependent upon itssuccess, are the numerous gene therapy approaches being advanced toinitial clinical trials that involve the ex vivo genetic manipulation ofcells and tissue. Gene therapy involves transduction of the genome ofthe cell to achieve correction of a defective gene, regulation of adisease condition, or production of a beneficial molecule. Those genetherapy procedures that will benefit from ex vivo administration of agene vector to an expanded or donor tissue in order to enhance thetargeting of the gene and avoid this systemic administration (likely toinclude most conceivable gene therapies for the next decade or longer)will be well served by the above advancements in tissue genesis andproduction.

The particular physical and biological requirements for the productionof cells and tissues of blood, skin, cartilage, bone, pancreas, thenervous system, and various other endothelial and mesenchymal tissues ofinterest to cell and genetic therapists, will vary. However, two keycomponents are necessary in order to grow cells and tissues ex vivo: 1)cells of self or donor origin that are capable of replicating anddifferentiating, as needed, for the formation of functioning tissue; and2) an ex vivo system comprised of biocompatible materials that providefor the physiological requirements (e.g., surface attachment, mediumexchange, and oxygenation) for the above cells to grow.

An excellent example of the merging interface of cell therapy withtissue engineering is the ex vivo production of human bone marrow. Thisprocess illustrates as well the interrelationship between thecell/tissue production methodology and the medical device requirementsto properly implement the tissue production.

Although lacking the physical geometry that is a feature of othertissues or organs, bone marrow is a tissue comprised of many differentcell types, ranging from different stromal fibroblasts, mesenchymalcells, to stem cells and the other cells of the hematopoietic system.The ex vivo process found to be needed for ex vivo bone marrow growth,was to mimic the natural functional environment of the bone marrow,providing for the controlled nutrient perfusion and oxygenation of thestem and stromal cell components under precise conditions of temperatureand medium composition. Key to the success was to provide cultureconditions that were concurrently amenable for each of the manydifferent cell types that are found in human bone marrow.

Using this approach of tissue engineering, for the first time, the humanstem cells that are found in the bone marrow were able to not onlysurvive in culture, but also replicate to produce more stem as well asmore mature progenitor cells. This result is in direct contrast to whenhematopoietic stem/progenitor cells have been isolated (e.g., CD-34selection) prior to the culture process. In this case the stem cells donot grow and the cultures die off over a short period, presumablybecause heterogeneous tissue interactions have been eliminated.

With the successful production of these bone marrow tissue cells, theycan be available to be used as a substitute for bone marrowtransplantation. This example is an excellent demonstration of how thelost function of a damaged or destroyed tissue, e.g., bone marrow, canbe repaired or restored with ex vivo engineered tissue-specific cells.

Once the basic cell/tissue production process is identified, the nextrequirement for therapeutic utilization is the need for clinical systemsto implement the process. These systems should be amenable for routineuse by the thousands of hospitals and clinics in the developed anddeveloping world that serve the patients intended to benefit from thetransplantation cells and tissues in native or genetically altered form.

2. Critical Requirements for an Ex-Vivo Cell Production Process

Cell and organ transplantation therapy to date has relied on theclinical facility to be able to handle and process cells or tissuesthrough the use of laboratory products and processes, governed tovarying degrees by standard operating procedures, and with varying FDAand other regulatory authority involvement. The procedures to date,however, generally have not required extensive manipulation of the cellsor tissue beyond providing standard incubation solutions, short termstorage or containment, or—as in the case of bone marrow or peripheralblood stem cells for stem cell transplants—cryopreservation. With theaddition of steps that require the actual growth and production of cellsor tissues for transplantation, there are many considerations that needto be addressed in order for a reliable and clinically safe process toresult. This issue is the same regardless of whether the cell productionis occurring at the patient care location (as might be the case for theproduction of cells for a stem cell transplant), or at some distantmanufacturing site (as might be the case for the production of abiosynthetic device).

A. Process Reliability for an Ex Vivo Cell Production Process

Perhaps the most critical of all issues to be addressed is the technicalart that is inherent with most cell culture processes. Site-to-sitedifferences in cell culture are often sensitive. For an acceptableclinical cell culture process, the technical art or sophistication mustbe eliminated such that the cell product will be the same when theprocess is used in different physical locations.

This problem can best be handled by implementing a well characterized,robust process with automation. While the variable human factor iseliminated from the technical process, human oversight should bemaintained for the quality monitoring and control process.Alternatively, highly controlled training and standard procedures can beused to address inherent variability in the practice of cell productionprocesses as well, and in certain cases will be required when thetechnical steps cannot be automated.

However, if a controlled process can lead to the same result repeatedly,then it can and should be automated. From a strategic point of view,automation via a medical device is desirable because it eliminatesvariability due to human error or human initiative, it reduces the needfor highly skilled labor and thus cost of the process, and it makes theprocess amenable for widespread practice. Ultimately, any manual processremains vulnerable to an ineffective automation strategy. Automationalso meets a general desire by clinicians to provide better qualityassurance to their patients.

B. Process Sterility—Closed Systems

With any cell culture procedure, a major concern is sterility. When theproduct cells are to be transplanted into patients—often at a time whenthe patient is immunocomprised, as is the case with stem celltransplants and with organ transplants—absence of microorganisms ismandated. Most laboratory cell culture procedures are carried out underaseptic conditions with the technician practicing so-called steriletechnique. Many of the bioreactor systems that have been developed offeradvantages over the manual processes in that once the culture isinitiated, the culture chamber and the fluid pathway is maintained in asterile, closed environment. However, even with these systems, theinitial setup and takedown steps, such as the medium priming andcollection of the cells at the completion of the process, requiresnon-sterile manual procedures.

Accordingly, laboratory cell culture systems are only partly closed,i.e., they involve numerous aseptic connections, are mostly operated ina controlled-environment hood, and have pre- and post-processing stepsrequiring open centrifuge tubes and the like. The most optimal objectiveis to have the culture process be carried out in a system where theculture chamber and fluid path is functionally closed to the externalenvironment, with the sterile integrity maintained from the time thedevice is manufactured until it has been disposed of.

C. Cell Recovery

For cell therapy, the product of the cell culture process is the cells.Accordingly, efficient collection of the cells at the completion of theculture process is an important feature of an effective cell culturesystem. Recovery of cells from most cell production processes is achallenge. They are either: 1) packed into the interstices of a make-dodialysis cartridge, or 2) suspended in many liters of culture medium.The first case necessitates unreasonable physical force to dislodgecells (being neither reliable nor easily automated), and the second caserequires significant time, patience, and a certain degree of goodfortune (being neither reliable nor closed).

The better approach for production of cells as the product is to culturecells in a defined, reasonable space, without physical barriers torecovery, so that simple elution of product results in a manageable,concentrated volume of cells amenable to final washing in a commercial,closed system cell washer designed for the purpose. An ideal systemwould allow for the efficient and complete removal of all cellsproduced, including both adherent and nonadherent cells. Furthermore,the harvest process should be able to be completed without breaking thesterile barrier of the fluid path of the culture chamber.

D. Optimization of Key Culture Parameters by Design

With any large volume cell culture, several fundamental parametersrequire almost constant control. Cultures must be provided with themedium that allows for normal metabolic functions and growth, andtypically this medium is delivered to the cell by a pumping mechanism(e.g., in a bioreactor) or by a technician manually feeding orexchanging the medium on a regular basis. As an additional part of thisexchange process, culture byproducts are also removed from the culture.

Growing cells or tissue also requires a source of oxygen. Different celltypes have different oxygen requirements, and all cell cultures havediffering oxygen delivery requirements depending on the density of theculture. Accordingly, a controllable and flexible means for providingoxygen to the cells is a necessary component of the culture system.

Even distribution of the cell population and medium supply in theculture chamber is an important process control. This control is oftenachieved by use of a suspension culture design, which can be effectivewhere cell-to-cell interactions are not as important as cell-to-mediuminteractions. Examples of suspension culture systems include varioustank reactor designs and gas-permeable plastic bags. Aside from matureblood cells, such as T-cells, such designs are often deleterious as theyimpede the development of three dimensional structure in tissue. Thegrowth of bone marrow stem cells is precluded in environments favoringsingle cell suspensions, because stem cells appear to desire contactwith stromal and other accessory cells in order to replicate.

E. Status Feedback and Production Record Capability

The essence of “good manufacturing practice” is: 1) equipment andfacilities that are capable of reliably providing the desired product,2) process control that through validation demonstrate an ability toproduce product within desired specifications, and 3) final product andprocess documentation controls that prevent mix-ups or inappropriaterelease of product before complete review of test and production recordsis conducted and accepted.

In a purely manual manufacturing environment, this is accomplished byselecting well-qualified personnel, providing them with extensivetraining, and developing a system of standard operating procedures andextensive documentation that provides for quality assurance. In anautomated manufacturing environment, the principles of processvalidation are used to demonstrate that the process is stable andcapable of meeting specifications. The principles of statistical processcontrol are then implemented to monitor the process to assure consistentconformance to specifications.

In today's environment, as cell culture becomes prominent in clinicalcare, much of the process control and record keeping will depend upontrained operators, maintaining detailed records. The future lies in theavailability of equipment and materials that are designed andmanufactured for the intended purpose and automation that allows theprocess to be validated and controlled. Only under these conditions cancellular therapy be delivered cost effectively to the market.

3. Cell Culture Devices and Procedures

In nature, tissue function and viability depends upon the life supportprocess that is mediated by the vascular system. Nutrients,physiological salts, and oxygen are all brought to the tissue througharteries. The waste products produced by the tissue, which can often betoxic to the tissue, are carried away by the veins. The other majorcomponent of tissue maintenance and repair is cellular—the pool ofprogenitor or stem cells that can replace cells lost or damaged.

Accordingly, for tissue to be developed ex vivo, these same elements arerequired to be managed by the culture devices and procedures. In otherwords, the stem/progenitor cells needed to expand the cellular componentof the tissue must be maintained in a physical and biologicalenvironment that is biocompatible and provides a means to controldelivery of nutrients and oxygen to the cells, and carry away waste orother byproducts of the growing cell populations.

A. Traditional Cell Culture Processes

Over the past decade, increasing numbers of medical researchers havesought to develop treatments based in part on the culture of human bloodcells, including lymphocytes, monocytes, neutrophil precursors, andimmature blood cells including stem and progenitor cells. The evolutionof technologies adapted or developed to meet these needs provides anexcellent demonstration of the need for clinical systems for theproduction of human cells for therapy.

1. Laboratory Environment

Traditional cell culture technologies depend upon controlledenvironments for cell handling. Cell culture laboratories incorporatesuch features as laminar flow hoods controlled access to the laboratoryby gowned personnel, and regular sterilization procedures todecontaminate laboratory surfaces. Personnel require extensive trainingto practice sterile technique, to avoid contamination of open containersand cell transfer devices by contact with non-sterile materials. Inspite of these prophylactic measures, outbreaks of contamination intraditional cell culture laboratories, e.g., fungus contamination, is acommon occurrence, often with the impact of halting operations for daysor weeks while the source of the contamination is determined andresolved.

Traditional cell culture technologies further depend upon incubation inan environment providing controlled gas mixtures and controlledtemperature, usually satisfied by the use of commercial incubatorsranging in size from large benchtop units to large floor-standing units.

Therapeutic requirements for numbers of blood cells (typically 10 to 100billion per patient treatment) and limitations in maximum cell culturedensity (typically one billion per liter of medium), together with spacerequirements for major laboratory hardware (e.g., hoods, incubators,refrigerators) and personnel activity, have resulted in considerablelaboratory space requirements per patient therapy. Laboratory supportoperations, including preparation of media and the practice of variousassays expand these space requirements and associated capitalinvestments and labor costs. Use of traditional cell culture technologyfor patient therapy thus results in relatively high costs per patienttreatment.

Such a laboratory environment is not conducive to the reliable androutine production of large numbers of cells for patient therapy, givenits reliance on manual, highly skilled technique. Achieving “goodmanufacturing practices” in such an environment is a daunting challenge,requiring the development and adherence to massive volumes of standardoperating procedures to eliminate inherent variability in laboratorypractices.

2. Tissue Culture Flasks and Roller Bottles

The earliest cell cultures were achieved in glass petri dishes, whichwere largely supplanted by pre-sterilized plastic tissue culture flasksin the 1960's and 1970's. Early attempts at large scale culture of humancells for therapy in the mid-1980's involved the use of numerous glassroller bottles in a room-sized mechanized facility. Even today, mostcell therapies have their genesis in plastic tissue culture flasks, andprocess scale-up involves use of more, larger so-called T-flasks, whichare fed manually in a laminar flow hood.

Manufacturers of tissue culture media used in human therapy havegradually moved away from glass bottle packaging to plastic bottles, andmost recently have been developing flexible plastic container systemsfor their media, similar to those used for decades for intravenous (IV)solutions. The use of flexible containers has been driven in part by thedesire by a few customers to eliminate open transfer steps for culturemedia, which can introduce potential contamination.

As described earlier, one preferred objective is to provide a cultureprocess that can deliver medium and oxygenation at uniform andcontrolled rates that mimic serum perfusion of tissue in vivo. In orderto achieve these relatively slow delivery rates, a means of internallyoxygenating the cells is often required. This is one requirement that isideally met using the simplest of cell culture processes—a culture dish.Here the surface of the culture is uniformly exposed to oxygen, andoxygen is available to the cells as needed. A similar situation can beproduced in a flatbed bioreactor, with a gas permeable/liquidimpermeable membrane a short distance from the cell bed. This allows fora system to have variable medium perfusion rates from stagnant to high,with the oxygenation of the culture remaining constant and uniform.

3. Flexible Tissue Culture Containers

Flexible tissue culture containers, or culture bags, were developed inthe mid-1980's, in response to clinicians' desire to perform culture ofcells for human therapy in a reproducible and reliable manner acrossmultiple laboratories and institutions. The use of aseptic tubingconnections technology, used commonly in the medical device industry(e.g., for blood collection and transfusion containers), rather thanconventional sterile technique in laminar flow hoods, reduces theprobability of contamination to less than one chance in a thousand perconnection. Flexible containers fitted with aseptic connectors wereappropriated from blood banking, where blood platelet concentrates werestored for several days in incubators in gas-permeable,liquid-impermeable plastic containers, which permitted bicarbonate pHbuffering of the platelets by the carbon dioxide gas in the incubator.

In the late 1980's, extensive trials of various forms of lymphocytetherapy were conducted using such culture bags, with low incidence ofcontamination. Today, these culture containers continue to find use inexperimental cell therapies where oxygen consumption requirements areminimal and non-adherent cells grow satisfactorily in suspensionculture. Such containers do not, however, support the growth of humanstem cells that require contact with a heterogeneous population ofadherent stromal cells. Advantages of culture bags include relativesimplicity of use, reduce skill level requirements, and potential usewithout laminar flow hoods. However, to date, processes utilizingculture bags remain labor- and space-intensive and are limited in theirclinical applicability.

4. Bioreactors

Platform-operated culture systems, typically referred to as bioreactors,have been available commercially for many years and employ a variety oftypes of culture technologies. Of the different bioreactors used formammalian cell culture, most have been designed to allow for theproduction of high density cultures of a single cell type. Typicalapplication of these high density systems is to produce as theend-product, a conditioned medium produced by the cells. This is thecase, for example, with hybridoma production of monoclonal antibodiesand with packaging cell lines for viral vector production. Theseapplications differ from applications where therapeutic end-product isthe harvested cells themselves.

These systems have made an important first step towards a usableclinical system. Once set up and running, the systems provideautomatically regulated (not necessarily uniform) medium flow, oxygendelivery, and temperature and pH controls, and they allow for productionof large numbers of cells. While bioreactors thus provide some economiesof labor and minimization of the potential for mid-processcontamination, the set-up and harvest procedures involve considerablelabor requirements and open processing steps, which require laminar flowhood operation (some bioreactors are sold as large benchtopenvironmental containment chambers to house the various individualcomponents that must be manually assembled and primed). Further, suchbioreactors are optimally designed for use with a homogeneous cellmixture, and not the mixture of cell types that exists with tissues suchas bone marrow.

Many bioreactors have a high medium flow rate requirement for operation.The reason of this feature is that the oxygenation mechanism is tooxygenate the medium outside of the growth chamber, immediately beforethe medium is perfused into the growth chamber. Since a high densityculture will quickly deplete the medium of oxygen, the medium must havea short residence time in the chamber, in order to be reoxygenated andrecirculated back into the culture chamber. Furthermore, this processresults in an absence of uniformity in oxygenation of the growingtissue, since cells proximal to the medium inlet see much higherconcentrations of oxygen than do the cells proximal to the mediumoutlet. This results in the different cells growing in different areasof the bioreactor.

An additional limitation is that many of the bioreactor designs, such asthe various three-dimensional matrix-based designs (e.g., hollow fibercartridges or porous ceramics), can impede the successful recovery ofexpanded cells and/or tissues, particularly when culture growth isvigorous, and also can limit mid-procedure access to cells for purposesof process monitoring.

The various trade-offs described have limited the utility of thesesystems and, in general, such bioreactors have not been used for humancell therapy as much as the less automated but often more user-friendlyculture bag systems.

It should, therefore, be appreciated that there is a need for a cellproduction system that can maintain and grow selected biological cellswithout being subject to the foregoing deficiencies. There is aparticular need for such a system that can receive, maintain and growsuch cells in a sterile system within a portable cassette withoutexposing that sterile system to the external environment. The presentinvention fulfills that need.

SUMMARY OF THE INVENTION

The present invention is embodied in an apparatus, and its componentsand related methods, that receives, maintains and grows biological cellsex vivo within a portable cassette, without exposing the cells to theexternal environment. The portable cassette includes a cell growthchamber within which the cells are maintained and grown, a mediacontainer configured to carry a quantity of a suitable growth medium,and a waste container configured to carry growth media discharged fromthe cell growth chamber. These elements are connected together to form asterile system that is closed to external environment. The closed,sterile system also can incorporate a harvest container configured tocarry growth media and biological cells discharged from the cell growthchamber.

In addition to the portable cassette, the apparatus further includes aplurality of instruments, each configured to receive and condition theportable cassette during a different stage of the cell growth process.The cassette is conveniently transportable from each instrument to thenext, as the cell growth process progresses. One such instrument is aprocessor configured for use in priming the cassette's cell growthchamber with growth media and in mixing biological cells with thatgrowth media and distributing the cells, e.g., uniformly, throughout thecell growth chamber. A second such instrument is an incubator configuredto condition the cassette while the biological cells are beingmaintained and grown. The processor instrument also is configured foruse in harvesting the biological cells after the incubation stage.

More particularly, the processor apparatus includes a support such as aplatform configured to removably receive the portable cassette andfurther configured to be movable in a controlled manner. A flow controlactuator is engageable with a media flow path of the portable cassettewhen the cassette is received by the support, and a controller controlsthe flow control actuator such that a determined quantity of the growthmedium is delivered from the media container to the cell growth chamberof the portable cassette. In addition, the controller thereaftercontrollably moves the support in a predetermined manner, such that thebiological cells are distributed substantially uniformly throughout thecell growth chamber. The portable cassette further includes a flowcontrol device engageable with the media flow path, and the flow controlactuator of the processor apparatus is engageable with that flow controldevice so as to control the delivery of growth media to the cell growthchamber. The controller includes a media flow sensor that senses thedelivery of growth media to the cell growth chamber and generates acorresponding detection signal, and the controller further is configuredto control the flow control device so as to regulate, e.g., terminate,the delivery of growth media.

The portable cassette can further include an inoculation port throughwhich a quantity of biological cells can be delivered to the cell growthchamber. In addition, the processor controller is configured to controlthe flow control actuator, after the inoculation of cells into the cellgrowth chamber, such that the delivery of growth media to the chamberterminates with a gas bubble remaining within the chamber. In addition,the processor controller is configured to then controllably move thesupport such that the gas bubble moves within the cell growth chamber ina predetermined manner, to mix the biological cells substantiallyuniformly with the growth media within the chamber. After that mixinghas been completed, the processor controller controls the flow controlactuator such that sufficient additional growth media is delivered tothe cell growth chamber to substantially displace the gas bubble.

In other features of the invention, the portable cassette includes twoseparate casings, including a first casing that defines the cell growthchamber and a second casing that defines the growth media container. Theincubator apparatus includes a first receptacle sized and configured toremovably receive the first casing, and a second receptacle sized andconfigured to removably receive the second casing. In addition, firstand second temperature regulators regulate the temperatures of the firstand second receptacles to prescribed temperatures, and an interface isprovided for engaging the portable cassette when the first and secondcasings are received in their respective receptacles, the interfacebeing configured to control the delivery of growth media from the mediacontainer to the cell growth chamber and to control the delivery of gasfrom a gas supply to the cell growth chamber. This provides theappropriate conditions for the biological cells to be maintained andgrown within the cell growth chamber.

The portable cassette includes a media flow path, e.g., plastic tubesand related connectors, in fluid communication with the cell growthchamber, e.g., between the chamber and the media container or betweenthe chamber and the waste container. Engaging this media flow path, andforming part of the portable cassette, is a flow control device, whichis configured to be engageable with a flow control actuator of aseparate instrument, e.g., the processor apparatus or the incubatorapparatus, and thereby control the flow of growth media through the cellgrowth chamber. The flow control device can include a plunger that isspring-biased to constrict the media flow path and a plate that isoperatively connected the plunger such that, when the plate isdepressed, the plunger releases its constriction of the flow path. Thisconstriction device conveniently can be mounted on the portable cassettewith the plate flush with a rear panel of the first casing.

The interface of the incubator apparatus includes an actuator engageablewith flow control device when the first casing of the portable cassetteis received in the first receptacle, and the actuator is controlled soas to deliver the growth media from the media container through the cellgrowth chamber to the waste container at a prescribed flow rate. Theinterface further includes a sensor that monitors the flow rate ofgrowth media being transported through the chamber.

In another feature of the invention, the processor apparatus further isconfigured to condition the portable cassette so as to harvestbiological cells that have been maintained within its cell growthchamber. This is achieved by initially controllably tilting the platformon which the cassette is received, while at the same time controllingone or more of various flow control devices, so that growth media andbiological cells within the chamber are discharged to the harvestcontainer via the harvest port. Thereafter, the processor apparatuscontrollably delivers a succession of reagents to the cell growthchamber, to dislodge additional cells from the cell bed and to deliverthose dislodged cells to the harvest container, again via the harvestport.

In a separate feature of the invention, the portable cassette includes amemory device that stores information about the portable cassette andits event history, the biological cells and growth media that arecarried by the cassette, and process instructions for the apparatus withwhich the cassette is configured to be used. The interfaces of both theprocessor apparatus and the incubator apparatus preliminarily retrieveinformation from this memory device and thereafter controllablycondition the cassette according to the retrieved information. Inaddition, the interfaces are configured to update the memory device withinformation pertinent to the condition of the portable cassette duringthe time it is received by respective apparatus. The memory deviceconveniently can be carried on the rear panel of the portable cassette'sfirst casing.

Other features and advantages of the present invention should becomeapparent from the following description of the preferred embodiment,taken in conjunction with the accompanying drawings, which illustrate,by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is schematic diagram of a cell production system in accordancewith the invention, including a portable cassette having a cell growthchamber, a processor instrument used to distribute inoculated biologicalcells within the cell growth chamber and later used to harvest the cellsthat have been maintained and grown, an incubator instrument forincubating the portable cassette such that the cells are maintained andgrown, and a system manager.

FIG. 2 is an exploded perspective view of the portable cassette of FIG.1.

FIG. 3 is a schematic diagram showing the interconnections between thecell growth chamber, the media container, the waste bag, the harvestbag, and the harvest reagent containers of the portable cassette of FIG.2.

FIG. 4 is an exploded perspective view of the cell growth chamber of theportable cassette of FIG. 2.

FIG. 5 is a perspective view of the rear wall of the portable cassetteof FIG. 2, showing the arrangement of fluid valves that control thedelivery of growth media and harvest reagents to the cassette's cellgrowth chamber, and further showing a drip tube used to monitor thegrowth media's flow rate through the chamber, and further showing thecassette's updatable memory device.

FIG. 6 is an exploded perspective view of one of several identical fluidcontrol valves included in the portable cassette of FIG. 2.

FIG. 7 is a perspective view of the processor instrument, which controlsthe portable cassette during both the cell inoculation and distributionstage, and the cell harvest stage, of the cell growth process.

FIG. 8 is a perspective view of the base portion of the processorinstrument, partially broken away, and showing the controllablyrotatable wheels and legs that support the tiltable platform.

FIG. 9 is a perspective view, partially broken away, of the interface ofthe incubator instrument, which controls the portable cassette duringthe incubation stage of the cell growth process.

FIG. 10 is a cross-sectional view of the heating module portion of theincubator instrument of FIG. 9, which is configured to receive the maincasing of the portable cassette.

FIG. 11 is a cross-sectional view of the cooling module portion of theincubator instrument of FIG. 9, which is configured to receive thesupplementary casing of the portable cassette.

DESCRIPTION OF THE PREFERRED EMBODIMENT

With reference now to the drawings, and particularly to FIGS. 1 and 2,there is shown a cell production system embodying the invention, for exvivo maintaining and growing biological cells such as human stem cellsand hematopoietic progenitor cells. The cells are grown from a smallstarting cell population, and, in the case of hematopoietic progenitorcells, a sufficient volume of cells can be grown and harvested tocomplete a bone marrow transplantation or a nadir prevention/rescueresulting from therapies such as high dose chemotherapy or radiation.The cell production system of FIG. 1 includes: 1) a disposable portablecassette (three cassettes 100A, 100B and 100C are depicted) having acell growth chamber (FIG. 2) in which the expansion and growth of thecells occurs, 2) a processor instrument 300 that is used initially toprime and inoculate the portable cassette and later to harvest cellsfrom the cassette, 3) an incubator instrument (two incubator instruments400A and 400B are depicted) that is used to control biological andphysical environment of the cell cassette while the cell expansion andgrowth is occurring, and 4) a system manager 500 that provides anoperator interface, for monitoring the cell growth occurringsimultaneously in as many as 50 separate portable cassettes.

As mentioned above, three separate portable cassettes are shown inFIG. 1. A first cassette 100A is shown by itself at the left of thedrawing, a second cassette 100B is shown received by the processorinstrument 300, and a third cassette 100C is shown received by theincubator instrument 400. Also as mentioned above, two separateincubator instruments are shown, one stacked above the other. The upperincubator instrument 400A is shown with its front door closed, and thelower incubator instrument 400B is shown with its front door removed, toreveal the instrument's reception of a portable cassette.

As shown in FIG. 2, the portable cassette 100 includes, in addition tothe cell growth chamber 102, a growth media container 104, a wastecontainer 106, and a harvest bag 108, all of which are connectedtogether to form a system that is closed to the external environment.The media container is initially charged with an appropriate growthmedium required for the cell culture, and it can include specifiedgrowth factors and glutamine. This system can be sterilized, as byirradiation, during the cassette's initial assembly. The processorinstrument 300 (FIG. 1) and the incubator instrument 400 both areconfigured to condition the portable cassette during separate stages ofthe cell growth process, without disturbing the cassette's closed,sterile system. Biological cells thereby can be received, maintained andgrown by the system without the need for highly trained laboratorypersonnel or special environmental equipment such as a laminar flowhood, but with minimal risk of contamination.

More particularly, and with reference to FIG. 2, the portable cassette100 includes a main casing 110 that houses the cell growth chamber 102,the waste container 106, and the harvest bag 108, and further includes asupplementary casing 112 that houses the growth media container 104(FIG. 3). As shown in FIG. 3, the cell growth chamber, in the preferredembodiment, is a shallow cylinder defined by a planar, disk-shaped,plastic cell bed 114 and an adjacent, closely spaced,gas-permeable/liquid-impermeable membrane 116. In use, biological cellsare distributed substantially uniformly over the cell bed, where theyare nourished by growth media that is pumped radially outwardly throughthe chamber at a selected flow rate. Oxygen and other prescribed gases,selected for pH stability, are supplied to the cells through themembrane 116, from a shallow, cylindrical gas chamber 118 located on theside of the membrane opposite the cell growth chamber.

With continued reference to FIGS. 2 and 3, the growth media is deliveredto the cell growth chamber 102 from the media container 104 via a mediasupply tube 120 and a supply port 122 located in the center of thechamber's cell bed 114. Waste media is delivered from the cell growthchamber to the waste container 106 via, in order, 1) a plurality ofports 123 (FIG. 4) spaced uniformly around the periphery of the cell bed114, 2) a shallow, cylindrical waste reservoir 124 located beneath thecell growth chamber, on the opposite side of the cell bed, 3) a wasteport 126 located near the center of that waste reservoir, and 4) a wastetube 128. Further, the biological cells that have been nourished andgrown in the cell growth chamber 102 are transferred to the harvest bag108 via a harvest port 130 located in the cell bed, adjacent to thechamber's periphery, and a harvest tube 132.

The media supply tube 120 and the waste tube 128 pass throughspring-biased valves 134 and 136, respectively, mounted on a rear panel138 of the main casing 110. The harvest tube 132 passes through asimilar spring-biased valve 140 mounted on a bottom wall 142 of the maincasing. These valves are appropriately controlled by the processorinstrument 300 and by the incubator instrument 400 during their separatestages of the cell growth process.

A portion of the waste tube 128 is configured as a drip chamber 144, foruse in measuring the flow rate of growth media moving through the tube,and thus through the cell growth chamber 102. The drip chamber includesa drip nozzle 146 that delivers the growth media in individual droplets,and this chamber is located adjacent to an aperture 148 in the rearpanel 138 of the main casing 110. Drip detectors 302 and 402 located inthe respective processor instrument 300 and incubator instrument 400 arepositioned and configured to automatically detect media droplets passingthrough the drip chamber when the corresponding instrument has received,and is conditioning, the portable cassette 100.

FIG. 6 is an exploded perspective view of the spring-biased media supplyvalve 134, which is used to control the flow of growth media through thesupply tube 120, from the media container 104 to the cell growth chamber102. This valve is identical in structure to the waste valve 136 and theharvest valve 140. The depicted valve includes a generally cylindricalbase 150 that is secured to the inner side of the main casing's rearpanel 138, aligned with a circular aperture (not visible in the drawing)formed in the panel. A cap 152 is attached to the inner end of the base,to define a central recess in which are located a plunger 156 and acompression spring 158 that biases the plunger toward the base.

The facing surfaces of the base 150 and the spring-biased plunger 156are configured as generally pyramidal anvils 160 and 162, respectively,and the media supply tube 120 is positioned between, and therebyconstricted by, the two anvils. The plunger includes a pair of legs 164a and 164 b that extend through slots (not shown) in the base and intothe aperture in the rear panel 138. A generally circular actuation plate168 is secured to the legs, and, when the valve is assembled, this platelies substantially flush with the rear panel. A space is defined beneaththe actuation plate, to accommodate movement of the plate toward of thebase, against the yielding bias of the compression spring 158. Thus, asufficient pressure applied to the actuation plate will overcome thespring bias and move the plunger's anvil 162 away from the base's anvil160, thereby removing the constriction of the media supply tube 120 andallowing growth media to flow through it.

With reference again to FIGS. 2, 3 and 5, oxygen and other prescribedgases are delivered to the portable cassette 100 via a gas-in connection172 located on the rear panel 138 of the main casing 110, and from thereare delivered to the gas chamber 118 via a gas-in line 174 and a gas-inport 176. Waste gases are discharged from the gas chamber via a gas-outport 178 and gas-out line 180 to a gas-out connection 182 located onthat same rear panel. These gas-in and gas-out connections are accessedby the incubator instrument 400 during the incubation stage of the cellgrowth process. The gas-in connection includes a sterile barrier filter183 to ensure that the gas delivered to the gas chamber remains sterile.The gas chamber preferably takes the form of a concentric circlelabyrinth, with the gas-in port located at one end of the labyrinth andthe gas-out port located at the other end.

The cell growth chamber 102 includes an enlarged center cavity 186extending above its center portion, which serves several importantfunctions, as will be described below. One such function is to providespace within the chamber to allow the inoculation of biological cellsthrough a septum 188, using a syringe 190. A vent, or center cavity,tube 192 extends from this center cavity through a spring-biased valve194 to a sterile vent 196. This valve is mounted on the rear panel 138of the main casing 110, and it has the same structure as the mediasupply valve 134, described above.

As mentioned above, the growth media container 104 is housed within thesupplementary casing 112. The media supply tube 120, which extends fromthe media container to the cell growth chamber 102, thus extends fromthe supplementary casing to the main casing 110. The growth mediacontainer preferably has a rigid body, and the growth media it containsis pressurized by an air supply line 200, which extends from thesupplementary casing to the main casing and terminates at an air supplyport 202 mounted on the main casing's rear panel 138. A sterile barrierfilter 203 ensures that the pressurized air that reaches the growthmedia is sterile. The air supply port 202 is accessed automatically bythe processor instrument 300 during the priming, inoculation anddistribution stage of the cell growth process, and by the incubationinstrument 400 during the incubation stage of the process.Pressurization of the growth media provides the pressure required totransport the media through the cell growth chamber 102 when the mediasupply valve 134 is opened.

A strain relief line or tether 204 interconnects the portable cassette'ssupplementary casing 112 and main casing 110. The line has a lengthslightly less than the lengths of the exposed media supply tube 120 andair supply line 200, so as to protect those elements from any tensilestresses brought about by an attempted movement of the two casingsexcessively apart from each other.

As shown specifically in FIG. 5, the portable cassette 100 furtherincludes a non-volatile, updatable memory device, or identification key,206 carried in a recess 208 in the cassette's rear panel 138. Thismemory device carries information about patient and the biological cellsbeing grown within the cassette's cell growth chamber 102, as well asprocess instructions for the instrument and information about thecondition of the cassette over time. The memory device is accessedautomatically by both the processor instrument 300 and the incubatorapparatus 400 when they receive the cassette, and the instrumentsretrieve the stored information and thereafter function to condition thecassette according to the retrieved information. Thus, the memory devicecan include detailed instructions relating to any desired program forconditioning the cassette during any particular stage of the cell growthprocess. Parameters such as media flow rate, gas mixture and flow rate,and temperature can be specified, and even can be made to vary overtime, to effect any desired program.

In addition, the processor instrument 300 and the incubator instrument400 are configured to update the memory device 206 with certaininformation about the condition of the portable cassette 100 during thetime periods in which they have received and are conditioning thecassette. Examples of the kind of information to be recorded are thecondition of the cassette at the occurrence of any interruption of theinstrument's operation, and the timing of that interruption. Thisfeature enables the cassette to be selectively removed from anyparticular instrument, as in the event of an instrument failure, andplaced in a substitute instrument for a continuation of the process, asthough no interruption had occurred.

One suitable memory device 206 for this purpose is available from DallasSemiconductor, of Dallas, Tex. It has the shape of a coin, and itsmemory contents can be retrieved and updated merely by physicallycontacting any portion of the device with a special read/write device,without the need for a special electrical connector or a criticalalignment of components. One example of such a read/write device is ahandheld wand 502 (FIG. 1), shown as part of the system manager 500.

With reference now to FIG. 7, there is shown the portion of theprocessor instrument 300 that receives and interfaces with the portablecassette 100 during the priming and cell inoculation and distributionstage, and later during the cell harvest stage, of the cell growthprocess. The processor instrument is shown to include a movable,generally rectangular platform 304 that is sized and configured toreceive and support the cassette's main casing 110 and further toinclude an overhead shelf 306 (FIG. 1) that is sized and configured toreceive and support the supplementary casing 112. Alternatively, theplatform could be enlarged so as to accommodate the supplementary casingalongside the main casing, in which case the two casings need not beseparable.

The processor instrument 300 further includes valve actuators 308, 310,312 and 314, which are positioned and configured to be automaticallyengageable with the respective spring-biased valves 134, 136, 140 and194 of the portable cassette 100, when the cassette's main casing 110 isproperly received on the platform 304. In the preferred embodiment, thevalve actuators each take the form of a simple solenoid having a plungerthat can be controllably biased outwardly to engage, and release, thecircular actuation plate of the associated spring-biased valve. Further,the processor instrument includes an air supply connector 315 that isconfigured to be automatically engageable with the cassette's air supplyport 202, when the cassette's main casing is so received, and aninternal valve (not shown in the drawings) that controllably suppliesair through this connector to pressurize the cassette's growth mediacontainer 104.

The processor instrument 300 further includes provisions on its overheadshelf 306 for supporting two reagent bags 214 and 216 that are a part ofthe portable cassette 100. As shown in FIGS. 1, 2 and 7 reagents inthese bags are used in the cell harvesting stage of the cell growthprocess, to rinse the cassette's cell growth chamber 102 and to dislodgeany biological cells adhered to the cell bed 114. The reagent bags 214and 216 are connected by tubes 218 and 220, respectively, and a commontube 222 to the cell growth chamber's enlarged center cavity 186. Thetubes 218 and 220 extend through first and second reagent valves 224 and226, respectively, which are located adjacent to the rear panel 138 ofthe cassette's main casing 110. These valves are identical inconstruction to the media supply valve 134, described above withreference to FIG. 6. The processor instrument further includes valveactuators 316 and 317 for controllably actuating the two reagent valves224 and 226, respectively, during the cell harvesting stage of the cellgrowth process, as will be described below.

As shown in FIG. 8, the platform 304 is supported on a main support leg318, which is located generally at the platform's center, and twoauxiliary support legs 320 a and 320 b, which are located near the rearcorners of the platform. The upper end of the main leg is secured to theplatform's underside by a universal joint 322, and the upper ends of theauxiliary legs 320 a and 320 b are secured to the platform's undersideby ball joints 324 a and 324 b, respectively. These joints allow limitedswiveling movement of the platform. The lower end of the main leg isfixed in a base portion 326 of the processor instrument 300, while thelower ends of the auxiliary legs 320 a and 320 b are secured to theperiphery of wheels 328 a and 328 b, respectively, which are mounted forcontrolled rotation in that base.

The wheels 328 a and 328 b both are controllably rotatable through 180degrees by stepper motors 330 a and 330 b, respectively, to move thelower ends of the legs 320 a and 320 b between predetermined lower andupper limits. By independently rotating the wheels in a synchronizedfashion, the platform 304, and thus the main casing 110 of the portablecassette 100, can be made to tilt in a controlled fashion. For example,positioning the wheels such that the lower ends of both auxiliary legsare at their highest positions will tilt the platform forward by amaximum amount, i.e., about 45 degrees in the preferred embodiment.Further, positioning the wheels such that the left-side auxiliary leg320 a is at its highest position and the right-side auxiliary leg 320 bis at its lowest position will tilt the platform to the right by amaximum amount. By controlling the wheel positions in an appropriatelysynchronized fashion, the platform can be made to move in a controlled,orbital motion, which is used in the preferred embodiment to distributethe inoculated biological cells substantially uniformly in thecassette's cell growth chamber 102. The platform's HOME position isdefined as the position at which it is substantially level and the lowerends of the legs are approximately at the midpoints of their verticaltravel.

During the priming and cell inoculation and distribution stage of thecell growth process, the valve actuators 308, 310, 312 and 314 areconditioned to controllably release the respective valves 134, 136, 140and 194, and the movable platform 304 is conditioned to controllablytilt, according to a predetermined operating sequence. This operatingsequence, which is discussed in detail below, is selected first to primethe cell growth chamber 102 with growth media delivered from the mediacontainer 104, then to facilitate inoculation of the chamber with thebiological cells to be maintained and grown, then to distribute thecells substantially uniformly throughout the chamber and thus uniformlyon the planar cell bed 114, and finally to completely fill the chamberwith growth media.

Table I sets forth the preferred operating sequence for the processorinstrument 300 during the priming and cell inoculation and distributionstage of the cell growth process for hematopoietic progenitor cells. Foreach step of the sequence, the Table sets forth a brief description ofthe function for the step, as well as the prescribed states for thevarious valves 134, 136, 140 and 194 of the portable cassette, 100, andfor several indicators 332 a, 332 b and 332 c on a front panel 334 ofthe processor instrument's base 336. The Table also sets forth, for eachstep of the sequence, the prescribed state for the pressurization valvethat supplies pressurized air to the cassette's growth media container104 and the prescribed position for the platform 304. The right-mostcolumns of the Table set forth the criteria for determining that thestep has been completed, and the time threshold for sounding an alarm ifeach particular step has not yet been completed.

In step 1 of the operating sequence, the processor instrument 300remains idle in its current condition while an operator places the maincasing 110 of the portable cassette 100 on the platform 304, whichautomatically engages the air supply connector 316 of the processorinstrument and positions the valves 134, 136, 140 and 194 adjacent tothe respective valve actuators 308, 310, 312 and 314. Proper placementof the main casing on the platform is sensed by a microswitch 338located on the platform. During this initial step, the operator alsoplaces the cassette's supplementary casing 112 on the overhead shelf 306(FIG. 1) and places the media supply tube 120 that interconnects the twocasings 110 and 112 in a tube sensor 340 mounted adjacent to theoverhead shelf.

When the operator has completed these connections, the processorinstrument 300 will illuminate the inoculate indicator 332 a, thecassette-in-place indicator 332 b, and the pause indicator 332 c, and itwill maintain closed all of the various valves of the portable cassette100 and the processor instrument. At this time, the operator can depressthe pause indicator 332 c, and the operating sequence will advance tostep 2. If the operator fails to depress this indicator within 300seconds, the instrument will sound an appropriate alarm.

When it is determined that the portable cassette 100 has been properlyreceived by the processor instrument 300, the instrument retrieves datastored in the memory device 206 that is conveniently located in therecess 208 in the rear panel 138 of the cassette's main casing 110. Thisdata includes an identification of the particular kind of biologicalcells to be maintained and grown, and the processor instrument selectsthe appropriate operating sequence based on this retrieved data.

In step 2 of the operating sequence for hematopoietic progenitor cellsof the kind to be maintained and grown in this example, the processorinstrument 300 moves the platform 304 to its HOME position, in which itis substantially level. This is accomplished by moving the wheels 328 aand 328 b to positions where the lower ends of the two auxiliary legs320 a and 320 b lie approximately midway between their extreme upper andlower positions. At this time, only the inoculate indicator 332 a andthe cassette-in-place indicator 332 b are illuminated and all of thevarious valves of the portable cassette 100 and processor instrumentremain closed. If the HOME position is not reached within 50 seconds, analarm is sounded; otherwise, the operating sequence will advance to step3.

In step 3, which is the first step of the priming sequence, theprocessor instrument 300 controllably tilts the platform 304 forward toan angle of about 9 degrees. This is accomplished by controllablyrotating the wheels 328 a and 328 b so as to raise the lower ends of theauxiliary legs 320 a and 320 b by appropriate amounts. During this time,the conditions of the indicators 332 a, 332 b and 332 c and of thevarious valves of the cassette 100 and processor instrument remainunchanged from their conditions during step 2. If the prescribed forwardtilt position is not reached within 10 seconds, an alarm is sounded;otherwise, the operating sequence will advance to step 4.

In step 4, the processor instrument 300 functions to introduce growthmedia into the cell growth chamber 102 of the portable cassette 100,from the growth media container 104. This is accomplished byconditioning the air supply valve to open, which pressurizes the mediacontainer, and at the same time by conditioning the valve actuator 308to controllably open the media supply valve 134, which allows the growthmedia to flow from the media container to the cell growth chamber viathe media supply tube 120 and the media supply port 122. At this time,the inoculate indicator 332 a and the cassette-in-place indicator 332 bremain illuminated.

As the growth media flows into the cell growth chamber 102, displacedair is vented through a vent tube 210, which connects the harvest tube132 (at a location upstream of the harvest valve 140) with the sterilevent 196. A Porex valve 212, which is located within this harvest tube,allows the venting air to pass through it unimpeded; however, as soon assufficient growth media has been introduced into the chamber to reachthe valve, the valve automatically closes and prevents the passage ofany further material. The Porex valve is available from a company calledPorex Technology, Inc. The automatic closing of the Porex valve causesan increase in the back pressure applied to the air supply pump locatedin the processor instrument 300, and sensing of this increased backpressure causes the operating sequence to proceed to step 5. If thisback pressure increase is not sensed to have occurred within 200seconds, an alarm is sounded.

In step 5, which is identical to step 2, the platform 304 is returned toits HOME position. If that operation is not completed within 50 seconds,an alarm is sounded. Otherwise, the program proceeds to step 6, in whichexcessive pressure within the cell growth chamber 102 of the portablecassette 100 is relieved by conditioning the valve actuator 314 to openthe vent valve 194. This allows any excessive air to be discharged fromthe cell growth chamber to the sterile vent 196 and the waste container106 via the waste reservoir 124, the waste port 126 and the waste tube128. Completion of this step is determined to have occurred when thedrip detector 302 fails to detect any drops of growth media through thedrip chamber 144 for about 5 seconds. When this occurs, the operatingsequence proceeds to step 7. On the other hand, if such a lack of dropshas not occurred within 20 seconds, an alarm is sounded.

Step 7 requires operator involvement, for the first time since theinitial placement of the portable cassette 100 on the processorinstrument 300. In this step, the platform 304 remains in its HOMEposition, and all three indicators 332 a, 332 b and 332 c areilluminated, with the inoculate indicator 332 a being made to flash. Inaddition, all of the various valve actuators are conditioned to maintaintheir corresponding valves closed. At this time, the operator isprompted to inject a quantity of biological cells into the cell growthchamber 102 via the septum 188 located in the chamber's enlarged centercavity 186. When the operator has completed his inoculation of cells, heis prompted to depress the pause indicator 332 c. In response, theprocessor instrument proceeds to step 8. If the operator fails todepress the pause indicator 332 c within 300 seconds, an alarm issounded.

Steps 8 through 15 all relate to the inoculation of the cell growthchamber 102 of the portable cassette 100 with the biological cells to bemaintained and grown, and to the distribution of those inoculated cellssubstantially uniformly throughout the chamber. In all of these steps,only the inoculate indicator 332 a and the cassette-in-place indicator332 b are illuminated, and all of the various valve actuators of thecassette and the processor instrument 300 are conditioned to maintaintheir associated valves closed.

In step 8, the processor instrument 300 tilts the platform 304, and thusthe main casing 110 of the portable cassette 100, rearward to an angleof 45 degrees. When this tilt angle has been reached, the sequenceadvances to step 9, in which air that previously had been trapped in theenlarged center cavity 186 of the cell growth chamber 102 is allowed tomigrate upwardly to the portion of the chamber that is tilted up. Abubble thereby is formed in this portion of the chamber. This step 9 hasa fixed duration of 30 seconds, after which the sequence automaticallyproceeds to step 10.

Step 10 is identical to steps 2 and 5, discussed above, i.e., theplatform 304 is conditioned to return to its HOME position. In the HOMEposition, the cell growth chamber 102 is substantially horizontal;however, surface tension of the growth media in the chamber prevents thebubble from migrating back to the enlarged center cavity 186. Even so,the operating sequence advances immediately to step 11 when the HOMEposition has been reached. If the HOME position has not been reachedwithin 30 seconds, an alarm is sounded.

In step 11, the platform 304 is made to tilt to an angle of about 25 to30 degrees and then to wobble in a controlled, step-wise, orbitalmotion. This motion moves the air bubble, which was formed in step 9,uniformly around the periphery of the cell growth chamber 102, and itthereby functions to distribute the inoculated cells throughout thechamber. This motion preferably has a cycle period of about 6 seconds,and it continues for about 120 seconds.

Steps 8-11 are substantially repeated in steps 12-15, but the wobblingoccurs for a reduced time interval and at a reduced tilt angle. Step 12tilts the platform 304 to an angle of about 45 degrees, and step 13holds the platform in that orientation for about 30 seconds, such that aclean air bubble again forms at the edge of the cell growth chamber'speriphery. Any small bubbles that broke away from the initial bubbleduring the wobbling of step 11 will consolidate during this step 13.Step 14 then returns the platform to its HOME position, and step 15 thentilts the platform to an angle of just 5 to 10 degrees and wobbles theplatform in a controlled, step-wise, orbital motion. This causes the airbubble to circle around the cell growth chamber 102 until it eventuallyreaches, and is captured by, the enlarged center cavity 186. This step15 has a duration of about 45 seconds.

Thereafter, in step 16, the air bubble that now is trapped in theenlarged center cavity 186 of the cell growth chamber 102 is purged.This is accomplished by introducing additional growth media into thechamber from the growth media container 104. In particular, the airsupply valve of the processor apparatus 300 is conditioned to open,which pressurizes the media container 104. At the same time, the valveactuator 308 is conditioned to controllably open the media supply valve134, which allows the growth media to flow from the media container tothe cell growth chamber via the media supply tube 120 and the mediasupply port 122. Pressure relief is provided by opening the vent valve194 in the tube 192 that connects the center cavity 186 with the dripchamber 144, which leads to the waste container 106. As soon as the dripdetector 302 has detected the occurrence of drops, it is determined thatthe cell growth chamber has been fully purged of air. In this step, onlythe inoculate indicator 332 a and the cassette-in-place indicator 332 bare illuminated.

Thereafter, in step 17, any excess pressure in the cell growth chamber102 is relieved by keeping open the vent valve 194, but closing thepressurization valve and the media supply valve 134. The conditions ofthe indicators 332 a, 332 b and 332 c remain unchanged. This step iscompleted when no drips have been detected for 5 seconds. When thisoccurs, the priming, inoculation and cell distribution stages of theprocess are determined to have been completed.

Finally, in step 18, the operator is prompted to remove the portablecassette 100 from the processor instrument 300. In particular, thecassette-in-place indicator 332 b is flashed, while the other twoindicator 332 a and 332 c remain dark, and all to the various valvesremain closed. The microswitch 338 detects removal of the cassette'smain casing portion 110 and flashing of the indicator lamp 332 b then isterminated. If the main casing is not removed within 300 seconds, analarm is sounded.

During the time that the portable cassette 100 is received by theprocessor instrument 300, the occurrence of any significant events isentered as data into the memory device 206 located on the cassette'srear panel 138. This is accomplished using a read/write device 341.Examples of data that is entered include the timing of the process andthe occurrence and timing of any alarms during the 18-step operatingsequence.

If the 18-step operating sequence is interrupted, e.g., by the operatordepressing the pause indicator 332 c, at any step, the processorinstrument 300 resumes implementing the sequence by proceeding through apredetermined recovery program. In particular, if the interrupt occursduring a step in which the platform 304 is to be moved to the HOMEposition (i.e., steps 2, 5, 10 and 14), then the instrument recoverssimply by moving the platform until that HOME position has been reached.If the interrupt occurs during a step in which the platform is toundergo some other movement, the instrument recovers by first returningthe platform to the HOME position and then repeating the step. On theother hand, if the interrupt occurs during a step in which the platformis not to be moved, then the instrument recovers simply by resuming thestep at the time when the interrupt occurred.

During the course of the 18-step priming, inoculation, and distributionoperating sequence, the contents of the memory device 206 are updated bythe read/write device 341 of the processor instrument 300 to reflectsuch information as: 1) the inoculation start and end times, 2) the stepnumber of the last step completed, 3) and an indication that cellinoculation and distribution has been completed. This updating ensuresthat, if the need ever arises, the portable cassette 100 can betransferred to a substitute processor instrument, at any step of thesequence, to complete the process.

It thus will be appreciated that the priming, cell inoculation, and celldistribution procedure is accomplished without breaking the sterilebarrier of the growth media container 104, the cell growth cassette 102,and the waste container 106. Moreover, it will be appreciated that thisprocedure is accomplished with only minimal operator involvement, andthat that minimal involvement does not require any sophisticatedoperator training.

After the priming, cell inoculation, and cell distribution procedure hasbeen accomplished, and the portable cassette 100 has been removed fromthe processor instrument 300, the cassette is in condition for cellincubation in the incubator instrument 400. In the case of hematopoieticprogenitor cells, the incubation procedure requires about two weeks. Inthat procedure, the cell growth chamber 102 is maintained at atemperature of about 37 degrees C., which provides optimal biologicalactivity for a culture, and the media container 104 is maintained at atemperature of about 4 degrees C., which minimizes breakdown ofheat-labile substances. At this same time, growth media is transportedthrough the cell growth chamber, from the media container to the wastecontainer 106, at a predetermined flow rate, and oxygen and other gasesare transported through the gas chamber 118 at a predetermined flowrate. These temperature and flow rate parameters are specified by theinformation stored in the memory device 206 located on the cassette'srear panel 138. In general the parameters are selected to provideoptimal growing conditions for the biological cells and, as mentionedabove, the parameters can be made to vary with time, according to anydesired program.

More particularly, and with reference to FIGS. 9-11, the incubatorinstrument 400 includes two side-by-side receptacles, i.e., a firstreceptacle 404 sized and configured to receive the main casing 110 ofthe portable cassette 100, and a second receptacle 406 sized andconfigured to receive the cassette's supplementary casing 112. The firstreceptacle 404 is configured to maintain the main casing 110, and thusthe cell growth chamber 102, at the prescribed 37-degree temperature,and it includes an appropriate interface to control the cassette's mediasupply valve 134 so as to deliver growth media at the prescribed flowrate(s) and to provide oxygen and other gases to the cassette's gaschamber 118. The second receptacle 406 is configured to maintain thesupplementary casing 112, and thus the media container 104, at theprescribed 4-degree C. temperature.

The first receptacle 404 of the incubator instrument 400 is sized andconfigured to slidably receive the main casing 110 of the portablecassette 100. Similarly, the instrument's second receptacle 406 is sizedand configured to slidably receive the cassette's supplementary casing112. The two receptacles are arranged in a side-by-side relationship,and a sealing front door 408 that spans the two receptacles can bepivoted closed after the two casings have been inserted into theirrespective receptacles, to seal the receptacles from the ambientenvironment. A microswitch 409 detects the proper insertion of the maincasing into the first receptacle.

The first receptacle 404 includes a conventional electrical heatingdevice 410 and associated fan 412, thermostat (not shown in thedrawings), and feedback control circuit (likewise, not shown), arrangedin a conventional manner, for maintaining the first receptacle'sinterior space at its prescribed temperature. Similarly, the secondreceptacle 406 includes a conventional thermoelectric cooling module 414and associated fan 416, thermostat (not shown), and feedback controlcircuit (not shown), for maintaining the second receptacle's interiorspace at its prescribed temperature.

A panel 418 at the rear of the first receptacle 404 carries a valveactuator 420 that is configured to be engageable with portablecassette's media supply valve 134, and it further carries the dripdetector 402, which is configured to be engageable with the cassette'sdrip chamber 144. A control system regulates the flow rate of growthmedia through the cassette's cell growth chamber 102 by controllablyactuating the valve actuator 420. The drip detector output iscontinuously monitored, to ensure that certain flow rate limits are notexceeded. Because the growth media does not serve as the oxygenationsource for the cell culture, the flow rate can be controlled to anextremely low level, which is considered more optimal for many cellexpansion applications.

An air supply connector 422 is configured to automatically mate with thecassette's air supply port 202, and pressurized air is selectivelydelivered to this connector from a suitable pump via an air supply valve(not shown). This selectively pressurizes the cassette's media container104 during the incubation process. In addition, a gas supply connector424 and a gas discharge connector 426 are positioned and configured toautomatically mate with the cassette's gas-in and gas-out connections172 and 182, respectively. This provides the desired gas mixture to thecassette's gas chamber 118 during the incubation process.

Also carried on the panel 418 at the rear of the first receptacle 404 isa read/write device 428 that can retrieve information from, and storeinformation to, the memory device 206 carried located on the rear panel138 of the portable cassette's main 110. When the cassette's main casingis properly inserted into the first receptacle, the read/write deviceretrieves the information stored in the memory device, to determine theprescribed temperature and time parameters for incubating the particularkind of cells to be grown. The incubator instrument 400 thenappropriately conditions its heating and cooling control systems,described above, to maintain the prescribed temperatures, and it alsothen starts a timer so that visible and audible alarms can be triggeredat appropriate times, as when the incubation procedure has beencompleted.

The read/write device 428 of the incubator instrument 400 also functionsto store information to the memory device 206. This informationpreferably can include: 1) the incubation start and end times, 2) theoccurrence and timing of any interruptions to the incubation procedure,such as alarms or power failures, 3) an identification of the amountgrowth media used, 4) an identification of the incubator instrument thatis used, and 5) an indication that incubation is complete. This updatingensures that, if the need ever arises, the portable cassette 100 can betransferred to a substitute incubator instrument, at any step of thesequence, to complete the process.

After the incubation stage of the cell growth process has beencompleted, the portable cassette 100 is returned to the processorinstrument 300, to harvest the cells from the cassette's cell growthchamber 102. As mentioned above, the processor instrument is configuredto implement the cell harvest stage of the process by conditioning thevalve actuators 312, 314, 316 and 317 to controllably release therespective harvest valve 140, center cavity valve 194, first reagentvalve 224, and second reagent valve 226, and by controllably tilting themovable platform 304, according to a predetermined operating sequence.This operating sequence, which is discussed in detail below, is selectedfirst to drain the cell growth chamber into the harvest bag 108, andthen to introduce several successive reagents into the chamber from thereagent bags 214 and 216, to ensure that substantially all of thebiological cells that have been grown are dislodged from the cell bed114, each time draining the reagent and dislodged cells into the harvestbag.

Table II sets forth the preferred operating sequence for the processorinstrument 300 during the cell harvest stage of the cell growth processfor hematopoietic progenitor cells. For each step of the sequence, theTable sets forth a brief description of the function for the step, aswell as the prescribed states for the various valves 140, 194, 224 and226 of the portable cassette 100, and for the several indicators 332 b,332 c and 332 d on the front panel 334 of the processor instrument'sbase 326. Table II also sets forth, for each step of the sequence, theprescribed position for the platform 304. The right-most columns ofTable II set forth the criteria for determining that the step has beencompleted and the time threshold for sounding an alarm if eachparticular step has not yet been completed.

In step 1 of the cell harvest operating sequence, the processorinstrument 300 remains idle in its current condition while an operatorplaces the main 110 of the portable cassette 100 on the platform 304,which automatically positions the valves 140, 194, 214 and 216 adjacentto the respective valve actuators 312, 314, 316 and 317. Properplacement of the main on the platform is sensed by the microswitch 338located on the platform. During this initial step, the operator alsoattaches the two harvest reagent bags 214 and 216 to an overhead support342, located adjacent to the instrument's overhead shelf 306. Aspreviously mentioned, the first bag 214 contains Hank's buffered salinesolution, and the second bag 216 contains Trypsin. The operator alsoplaces the first and second reagent tubes 218 and 220 in tube sensors344 and 346, respectively, located on the overhead support. Theprocessor instrument 300 thereby senses that the harvesting operatingsequence can be initiated.

When the operator has completed these connections, the processorinstrument 300 will illuminate the cassette-in-place indicator 332 b,the pause indicator 332 c, and the harvest indicator 332 d, and it willmaintain closed all of the various valves of the portable cassette 100.At this time, the operator can depress the pause indicator 332 c, andthe operating sequence will advance to step 2. When that has occurred,the pause indicator 332 c will darken; however, the cassette-in-placeindicator 332 b and the harvest indicator 332 d will remain lit for theremainder of the harvesting stage. If the operator fails to depress thisindicator within 300 seconds, the instrument will sound an appropriatealarm.

When it is determined that the portable cassette 100 has been properlyreceived by the processor instrument 300, the instrument retrieves datastored in the memory device 206 that is conveniently located in therecess 208 located in the rear panel 138 of the cassette's main casing110. This is accomplished using the read/write device 341. The retrieveddata includes an identification of the particular kind of biologicalcells that have been grown and the appropriate procedure for harvestingthose cells, and the processor instrument then selects the appropriateoperating sequence based on this retrieved data. The operating sequenceset forth in Table II is appropriate for harvesting hematopoietieprogenitor cells.

In step 2 of the operating sequence for harvesting hematopoieticprogenitor cells of the kind that have been grown in this example, theprocessor instrument 300 moves the platform 304 to its HOME position, inwhich it is substantially level. At this time, all of the various valvesof the portable cassette 100 remain closed. The cassette-in-placeindicator 332 b and the harvest indicator 332 d remain illuminated, asthey will for the entire operating sequence of the harvest stage. If theHOME position is not reached within 50 seconds, an alarm is sounded;otherwise, the operating sequence will advance to step 3.

Steps 3-8 all relate to the initial draining of fluid from the cellgrowth chamber 102 into the harvest bag 108. The fluid that is initiallydrained includes the growth media and a major portion of the biologicalcells that have been grown. The remaining portion of the cells remainadhered to the cell bed 114.

In step 3, the processor instrument 300 tilts its platform 304, and thusthe portable cassette 100, rearward to an angle of about 45 degrees andit opens the center port valve 194, to vent the cell growth chamber 102to the atmosphere via the vent tube 192 and the sterile vent 196. Afterthis has been done, the process proceeds to step 4, in which the harvestvalve 140 is opened. This drains most of the aforementioned fluid fromthe cell growth chamber into the harvest bag 108, via the harvest port130 and the harvest tube 132. This step has a duration of 105 seconds.

Thereafter, in step 5, the platform 304 is returned to its HOME positionand, in step 6, the platform is again tilted rearward to an angle ofabout 45 degrees. Step 7 holds the platform in this tilted position fora duration of 60 seconds, while an additional amount of the fluid isdrained into the harvest bag 108. The harvest valve 140 and the centerport valve 194 remain open during these steps 5-7. In step 8, theharvest valve is closed and the platform is returned to its HOMEposition.

Steps 9-16 all relate to the rinsing of the cell growth chamber 102 withthe reagent contained in the first reagent bag 214, i.e., Hanks bufferedsaline solution, and the draining of the resulting rinse solution intothe harvest bag 108. In step 9, the valve 224 for the first reagent isopened, and about 70 ml. of the reagent is transferred into the cellgrowth chamber. This represents about one half of the total amount ofthis reagent initially carried in the bag 214. Thereafter, in step 10,the first reagent valve 224 is closed and the center port valve 194 isopened, to vent the chamber, and the platform 304 is made to oscillatein the forward and rearward direction. This oscillation has a range ofabout +45 degrees to about −45 degrees, and it moves the reagentrepeatedly across the cell bed 114, to dislodge a portion of the adheredcells. This motion continues for 60 seconds.

The platform 304 then returns to its HOME position, in step 11, and ismade to oscillate side-to-side, in step 12. This motion again has arange of about +45 degrees to about −45 degrees and a duration of 60seconds, and it dislodges yet further adhered cells. Thereafter, theplatform returns to its HOME position, in step 13, and then tiltsrearward to an angle of about 45 degrees, in step 14. The harvest valve140 then is opened, in step 15, and the reagent and dislodged cells arethereby drained into the harvest bag 108. This step 15 has a duration of75 seconds. Finally, in step 16, the harvest valve is closed and theplatform is returned to its HOME position.

Steps 17-24 all relate to the rinsing of the cell growth chamber 102with the reagent carried in the second reagent bag 216, i.e., Trypsin,and the draining of the resulting rinse solution into the harvest bag108. Trypsin is a protein that is effective in dislodging nearly all ofthe remaining cells adhered to the cell bed 114. The steps 17-24 areidentical to steps 9-16, discussed above, except that step 17 opens thevalve 226 for the second reagent, which contrasts with step 9's openingof the valve 216 for the first reagent. After the final step, i.e., step24, the platform 304 is in its HOME position.

Steps 25-32 all relate to a second rinsing of the cell growth chamber102 with the reagent (Hank's buffered saline solution) carried in thefirst reagent bag 214. These steps are identical to steps 9-16,discussed above, and they serve to transfer to the harvest bag 108 mostof any the cells remaining in the chamber. After step 32, the platform304 is in its HOME position.

Finally, in step 33, all of the valves of the portable cassette 100 andthe processor instrument 300 are closed and the cassette-in-placeindicator 332 b is made to flash. In addition, an audible prompt isprovided, to prompt the operator to remove the cassette from theinstrument and to extract the harvest bag 108. At this time, the harvestbag should carry substantially all of the biological cells present inthe cell growth chamber 102 when the harvesting procedure was initiated,as well as the growth media then present in the chamber, about 140 ml.of Hank's buffered saline solution, and about 70 ml. of Trypsin.

If the 33-step operating sequence of the harvesting procedure isinterrupted at any step, e.g., by the operator depressing the pauseindicator 332 c, the processor instrument 300 resumes implementing thesequence by proceeding through a predetermined recovery program. Inparticular, if the interrupt occurs during a step in which the platform304 is to be moved to the HOME position (i.e., steps 2, 5, 8, 11, 13,16, 19, 21, 24, 27, 29 and 32), then the instrument recovers simply bymoving the platform until that HOME position has been reached. If theinterrupt occurs during a step in which the platform is to undergo someother movement, the instrument recovers by first returning the platformto the HOME position and then repeating the step. On the other hand, ifthe interrupt occurs during a step in which the platform is not to bemoved, then the instrument recovers simply by resuming the step at thetime when the interrupt occurred.

While the portable cassette 100 is received by the processor instrument300, the occurrence of any significant events is entered as data by theread/write device 341 into the memory device 206 located on thecassette's rear panel 138. Examples of such data include: 1) the harveststart and end times, 2) an identification of the last operating stepcompleted, 3) the occurrence and timing of any alarms during its 33-stepoperating sequence, and 4) an indication that cell harvesting has beencompleted. This updating ensures that, if the need ever arises, theportable cassette 100 can be transferred to a substitute processorinstrument, at any step of the sequence, to complete the process.

It thus will be appreciated that the cell harvesting procedure isaccomplished without breaking the sterile barrier of the cell growthcassette 102 and the harvest bag 108. Moreover, it will be appreciatedthat this procedure is accomplished with only minimal operatorinvolvement, and that that minimal involvement does not require anysophisticated operator training.

As mentioned above, the system manager 500 (FIG. 1) provides an operatorinterface and monitors the cell growth occurring simultaneously in asmany as 50 separate portable cassettes 100. The system manager isnetworked to a plurality of processor instruments 300 and a plurality ofincubator instruments 400, to monitor the status of those instrumentsand any portable cassettes they might at any time be processing. Nodirect control of those instruments by the system manager occurs;rather, the system manager's only function in this regard is to monitorthe instruments and to amass information pertinent to the condition ofeach of the cassettes during the course of its cell growth.

Examples of the kind of information that the system manager 500 canreceive from any particular processor instrument 300 include: 1) theinstrument's status and the particular step number it might currently beimplementing, 2) the settings of the valves of the portable cassette 100being processed, 3) the readings of the instrument's various sensors, 4)the information stored in the memory device 206 of the associatedportable cassette, 5) the status of any alarms, and 6) an identificationof the particular operating sequence being implemented. Examples of thekind of information that the system manager can receive from anyparticular incubator instrument 400 include: 1) the instrument's statusand its control settings, 2) the readings of the instrument's varioussensors, 3) the information stored in the memory device 206 of theassociated portable cassette, 4) the status of any alarms, and 5) anidentification of the particular operating sequence being implemented. Aprinter 503 can print this information at the end of the cell growthprocess, or on command, to provide a written record of the process, forarchival purposes.

Another function of the system manager 500 is to initially loadpertinent information into the memory device 206 associated with eachportable cassette 100, before the cell growth process is begun. Suchinformation can include: 1) an identification of the particular patientfor whom the cells are to be grown, 2) an identification of the type ofcells to be grown, 3) an identification of the particular portablecassette being used and the particular lots of growth media and harvestreagents being used, 4) a real time and date stamp, and 5) anidentification of any particular processing parameters (e.g., incubationtemperature and duration) for the cell growth process. To this end, thesystem manager includes a keyboard 504 for manual data entry and a barcode reader 506 for scanning bar code labels on reagent bags and thelike. The system manager also includes the read/write device 502, forloading the information into the memory device.

The system manager 500 also can be used to check the memory device 206after incubation, but before cell harvesting. This check can ensure thatthe portable cassette 100 is, in fact, ready to undergo the cellharvesting procedure.

It should be appreciated from the foregoing description that the presentinvention provides an apparatus, and related method, that receives,maintains and grows biological cells ex vivo within a portable cassette,without exposing the cells to the external environment. The portablecassette is used in combination with a processor instrument thatinoculates the cassette with cells of the kind to be grown anddistributes those cells in a predetermined pattern (e.g., uniformly)throughout a cell growth chamber, and thereafter in combination with anincubator instrument that incubates the cell growth chamber so that thecells are optimally expanded. The same processor instrument then is usedto harvest the expanded cells from the portable cassette. Bothinstruments are configured to condition the portable cassette duringstages of the cell growth process, without disturbing the cassette'ssterile system. In addition an updatable memory device associated withthe cassette stores significant information about the cassette and itscondition during the various steps of the cell growth process. Suchinformation is useful both for subsequent archival purposes and forfacilitating a resumption of the cell growth process in the event of anyinstrument failure or significant alarm condition.

TABLE I Processor Sequencing Chart - Inoculate Procedure Alert/AlarmChecks Step done Motion Inoculate Cassette-in-place Pause B-MediaD-Harvest E-Center F-Harvest G-Harvest Pressurization Timeout TimeoutStep Description Indicator Indicator Indicator Supply C-Waste Bag PortReag. #1 Reag. #2 Valve (seconds) (seconds) [Idle to Run transition,self-tests] 1 Programmed pause for container Lit Lit Lit C C C C C C CKey Press 300 — set up 2 Go to home Lit Dark C C C C C C C Program Done50 — 3 Tilt cassette to prime angle Lit Lit Dark C C C C C C C ProgramDone 10 — 4 Fill cassette w/liquid Lit Lit Dark O C C C C C O PressureRise to 200 — Threshold 5 Go to home Lit Lit Dark C C C C C C C ProgramDone 50 — 6 Relieve excess cell bed pressure Lit Lit Dark C O C C C C CNo Drips for 5 sec 20 — 7 Programmed pause for innoculum Flash Lit Lit CC C C C C C Key press 300 — addition 8 Tilt cassette to +45° Lit LitDark C C C C C C C Program Done 10 — 9 Wait for Bubble to form Lit LitDark C C C C C C C 30 seconds — — 10 Go to Home Lit Lit Dark C C C C C CC Program Done 50 — 11 Wobbulate to distribute cells Lit Lit Dark C C CC C C C Program Done 180 10 12 Tilt cassette to +45° Lit Lit Dark C C CC C C C Program Done 10 — 13 Wait for Bubble to form Lit Lit Dark C C CC C C C 30 seconds — — 14 Go to Home Lit Lit Dark C C C C C C C ProgramDone 45 10 15 Wobbulate to center bubble Lit Lit Dark C C C C C C CProgram Done 45 10 16 Purge center port Lit Lit Dark O C C O C C O Dripdetection 10 — 17 Relieve cell bed pressure Lit Lit Dark C C C O C C CNo Drips for 5 sec 20 — 18 Remove cell cassette for incubation DarkFlash Dark C C C C C C C DBC Microswitch open 300 — [Return to IdleState] C = Valve closed (Off) O = Valve open (On) For alert/alarmcolumns, bold entry = alert, and non-bold entry = alarm

TABLE II Processor Sequencing Chart - Harvest Procedure Alert/AlarmChecks Step Down Motion Harvest Cassette-in-place Pause B-MediaD-Harvest E-Center F-Harvest G-Harvest Power Failure/ Timeout TimeoutStep Description Indicator Indicator Indicator Supply C-Waste Bag PortReag. #1 Reag #2 Pause Recovery Criteria (seconds) (seconds) 21 Go tohome Lit Lit Dark C C C O C C Sequence 7 Program Done 50 — 22 TiltCassette to +45 Lit Lit Dark C C C O C C Sequence 7, Program Done 10 —Repeat Step 23 Drain cell bed into harvest bag Lit Lit Dark C C O O C CResume exactly 75 seconds — — where left off 24 Go to home Lit Lit DarkC C C O C C Sequence 7 Program Done 50 — 25 Add rest of Reagent #1 tocell bed Lit Lit Dark C C C C C O Resume exactly 40 seconds — — whereleft off 26 X axis wash Lit Lit Dark C C C O C C Sequence 7, 60 seconds— 10 Resume at time left 27 Go to home Lit Lit Dark C C C O C C Sequence7 Program Done 50 — 28 Y axis wash Lit Lit Dark C C C O C C Sequence 7,60 seconds — 10 Resume at time left 29 Go to home Lit Lit Dark C C C O CC Sequence 7 Program Done 50 — 30 Tilt Cassette to +45 Lit Lit Dark C CC O C C Sequence 7, Program Done 10 — Repeat Step 31 Drain cell bed intoharvest bag Lit Lit Dark C C O O C C Resume exactly 90 seconds — — whereleft off 32 Go to home Lit Lit Dark C C C O C C Sequence 7 Program Done50 — 33 Recover Harvest Bag, Remove Cassette Dark Flash Dark C C C C C CResume exactly Microswitch Open 300 — where left off [Return to IdleState] C = Valve closed (Off) O = Valve open (On) For alert/alarmcolumns, bold entry = alert, and non-bold entry = alarm

Although the invention has been described in detail with reference onlyto the presently preferred embodiment, those skilled in the art willappreciate that various modifications can be made without departing fromthe invention. Accordingly, the invention is defined only by thefollowing claims.

What is claimed is:
 1. Apparatus for ex vivo maintaining and growingbiological cells, comprising: a portable cell growth cassette thatincludes a casing a defines a cell growth chamber configured to carry aquantity of biological cells and a growth medium, a media containerconfigured to carry a growth medium, and a waste container configured tocarry media discharged from the growth chamber, wherein the cell growthchamber, the media container, and the waste container are connectedtogether to form a sterile system that is closed to the externalenvironment; a first instrument, separate from the portable cell growthcassette, that includes a mechanical interface to which the cassette canbe removably coupled, the first instrument thereupon controllablyconditioning the cassette during a first stage of its ex vivomaintenance and growth of biological cells, without exposing the closed,sterile system to the external environment wherein such controllableconditioning includes controllably transporting growth media through thecell growth chamber; and a second instrument, separate from the portablecell growth cassette, that includes a mechanical interface to which thecassette can be removably coupled, the second instrument thereuponcontrollably conditioning the cassette during a second stage of its axvivo maintenance and growth of biological cells, without exposing theclosed, sterile system to the external environment wherein suchcontrollable conditioning includes controllably transporting growthmedia through the cell growth chamber.
 2. Apparatus as defined in claim1, wherein: the first instrument is configured to controllably conditionthe portable cassette such that growth media and inoculated biologicalcells are distributed within its cell growth chamber, without exposingthe closed, sterile system to the external environment; and the secondinstrument is configured to controllably incubate the portable cassettesuch that the biological cells are maintained and grown within the cellgrowth chamber, without exposing the closed, sterile system to theexternal environment.
 3. Apparatus as defined in claim 2, wherein thefirst instrument further is configured to controllably condition theportable cassette such that biological cells maintained and grown withinthe cell growth chamber are harvested, without exposing the closed,sterile system to the external environment.
 4. Apparatus as defined inclaim 2, wherein the second instrument further is configured tocondition the portable cassette by controlling the flow of growth mediafrom the media container through the cell growth chamber to the wastecontainer, and by providing a flow of a gas containing oxygen to thecell growth chamber and a flow of discharge gases from the cell growthchamber.
 5. Apparatus as defined in claim 1, wherein: the portablecassette further includes a memory device that carries information aboutthe cell growth chamber and about the desired conditioning of the cellgrowth chamber over time; and the first and second instruments both areconfigured to retrieve information from the memory device of theportable cassette and to condition the cassette based on the retrievedinformation.
 6. Apparatus as defined in claim 1, wherein: the portablecassette further includes an updatable memory device; and the first andsecond instruments both are configured to store information to thememory device of the portable cassette based on the condition of thecassette during the time in which that instrument has received it.
 7. Amethod for ex vivo maintaining and growing biological cells, comprising:providing a portable cell growth cassette that includes a casing thatdefines a cell growth chamber configured to carry a quantity ofbiological cells and a growth medium, a media container connected to thecell growth chamber and configured to carry a growth medium, and a wastecontainer connected to the cell growth chamber and configured to carrymedia discharged from the growth chamber, wherein the cell growthchamber, the media container, and the waste container form a sterilesystem that is closed to the external environment; positioning theportable cell growth cassette on a platform of a first instrument,separate from the portable cassette, wherein positioning includescoupling the cassette with a mechanical interface that is part of thefirst instrument; and conditioning the portable cassette while it ispositioned on the first instrument platform so as to implement apredetermined stage of a process for ex vivo maintaining and growingbiological cells within the cell growth chamber, without exposing theclosed, sterile system to the external environment, wherein conditioningincludes controllably transporting growth media through the cell growthchamber.
 8. A method as defined in claim 7, and further including:positioning the portable cell growth cassette on a platform of a secondinstrument, separate from the portable cassette, wherein positioningincludes coupling the cassette with a mechanical interface that is partof the second instrument; and conditioning the portable cassette whileit is positioned on the second instrument platform so as to implement asecond predetermined stage of the process for ex vivo maintaining andgrowing biological cells, without exposing the closed, sterile system tothe external environment, wherein conditioning includes controllablytransporting growth media through the cell growth chamber.
 9. A methodas defined in claim 8, wherein: conditioning the portable cassette whileit is positioned on the first instrument platform distributes growthmedia and inoculated biological cells within the cell growth chamber,without exposing the closed, sterile system to the external environment;and conditioning the portable cassette while it is positioned on thesecond instrument platform incubates the portable cassette such that thebiological cells are maintained and grown within the cell growthchamber, without exposing the closed, sterile system to the externalenvironment.
 10. A method as defied in claim 9, and further including:again positioning the portable cassette on the platform of the firstinstrument, wherein again positioning includes coupling the cassettewith the mechanical interface of the first instrument; and conditioningthe portable cassette while it is again positioned on the firstinstrument platform such that biological cells maintained and grownwithin the cell growth chamber are harvested, without exposing theclosed, sterile system to the eternal environment.
 11. A method asdefined in claim 9, wherein conditioning the portable cassette while itis positioned on the second instrument platform includes: controllingthe flow of growth media from the media container through the cellgrowth chamber to the waste container; and providing a flow of a gascontaining oxygen to the cell growth chamber and a flow of dischargegases from the cell growth chamber.
 12. A method as defined in claim 7,wherein: the portable cassette further includes a memory device thatcarries information relating to the desired conditioning of the cellgrowth chamber over time; and conditioning the portable cassette whileit is positioned on the first instrument platform includes retrievinginformation from the memory device of the portable cassette andconditioning the cassette based on the retrieved information.
 13. Amethod as defined in claim 7, wherein: the portable cassette furtherincludes an updatable memory device; and conditioning the portablecassette while it is positioned on the first instrument platformincludes storing information to the memory device of the portablecassette, such information relating to the condition of the cassettewhile it is so positioned.
 14. Apparatus for ex vivo maintaining andgrowing biological cells, comprising: a portable cell growth cassettethat includes a casing that defines a cell growth chamber configured tocarry a quantity of biological cells and a growth medium, the cellgrowth chamber being part of a sterile system that is closed to theexternal environment; and a first instrument, separate from the portablecell growth cassette, that includes a mechanical interface to which thecassette can be removably coupled, the first instrument thereuponcontrollably conditioning the cassette during a first stage of its exvivo maintenance and growth of biological cells, without exposing theclosed, sterile system to the external environment wherein suchcontrollable conditioning includes controllably transporting growthmedia through the cell growth chamber.
 15. Apparatus as defined in claim14, and further including a second instrument, separate from theportable cell growth cassette, that includes a mechanical interface towhich the cassette can be removably coupled, the second instrumentthereupon controllably conditioning the cassette during a second stageof its ex vivo maintenance and growth of biological cells, withoutexposing the closed, sterile system to the external environment, whereinsuch controllable conditioning includes controllably transporting growthmedia through the cell growth chamber.
 16. Apparatus as defined in claim15, wherein: the first instrument is configured to controllablycondition the portable cassette such that growth media and inoculatedbiological cells are distributed within its cell growth chamber, withoutexposing the closed, sterile system to the external environment; and thesecond instrument is configured to controllably incubate the portablecassette such that the biological cells are maintained and grown withinthe cell growth chamber, without exposing the closed, sterile system tothe external environment.
 17. Apparatus as defined in claim 16, whereinthe first instrument further is configured to controllably condition theportable cassette such that biological cells maintained and grown withinthe cell growth chamber are harvested, without exposing the closed,sterile system to the external environment.
 18. Apparatus as defined inclaim 16, wherein: the portable cell growth cassette further includes amedia container configured to carry a growth medium, and a wastecontainer configured to carry media discharged from the growth chamber;the cell growth chamber, the media container, and the waste containerare connected together to form a sterile fluid pathway that constitutesthe sterile system that is closed to the external environment; and thesecond instrument further is configured to condition the portablecassette by controlling the flow of growth media from the mediacontainer through the cell growth chamber to the waste container, and byproviding a flow of a gas containing oxygen to the cell growth chamberand a flow of discharge gases from the cell growth chamber, withoutexposing the sterile fluid pathway to the external environment. 19.Apparatus as defined in claim 14, wherein: the portable cassette furtherincludes a memory device that carries information about the cell growthchamber and about the desired conditioning of the cell growth chamberover time; and the first instrument is configured to retrieveinformation from the memory device of the portable cassette and tocondition the cassette based on the retrieved information.
 20. Apparatusas defined in claim 14, wherein: the portable cassette further includesan updatable memory device; and the first instrument is configured tostore information to the memory device of the portable cassette based onthe condition of the cassette during the time in which that instrumenthas received it.
 21. A method for ex vivo maintaining and growingbiological cells, comprising: providing a portable cell growth cassettethat includes a casing that defines a cell growth chamber configured tocarry a quantity of biological cells and a growth medium, wherein thecell growth chamber is part of a sterile system that is closed to theexternal environment; positioning the portable cell growth cassette on aplatform of a first instrument, separate from the portable cassette,wherein positioning includes coupling the cassette with a mechanicalinterface that is part of the first instrument; and conditioning theportable cassette while it is positioned on the first instrumentplatform so as to implement a first predetermined stage of a process forex vivo maintaining and growing biological cells within the cell growthchamber, without exposing the closed, sterile system to the externalenvironment wherein such conditioning includes controllably transportinggrowth media through the cell growth chamber.
 22. A method as defined inclaim 21, and further including: positioning the portable cell growthcassette on a platform of a second instrument, separate from theportable cassette, wherein positioning includes coupling the cassettewith a mechanical interface that is part of the second instrument; andconditioning the portable cassette while it is positioned on the secondinstrument platform so as to implement a second predetermined stage ofthe process for ex vivo maintaining and growing biological cells,without exposing the closed, sterile system to the external environment,wherein such conditioning includes controllably transporting growth. 23.A method as defined in claim 22, wherein: conditioning the portablecassette while it is positioned on the first instrument platformdistributes growth media and inoculated biological cells within the cellgrowth chamber, without exposing the closed, sterile system to theexternal environment; and conditioning the portable cassette while it ispositioned on the second instrument platform incubates the portablecassette such that the biological cells are maintained and grown withinthe cell growth chamber, without exposing the closed, sterile system tothe external environment.
 24. A method as defined in claim 23, andfurther including: again positioning the portable cassette on theplatform of the first instrument, wherein again positioning includescoupling the cassette with the mechanical interface of the firstinstrument; and conditioning the portable cassette while it is againpositioned on the first instrument platform such that biological cellsmaintained and grown within the cell growth chamber are harvested,without exposing the closed, sterile system to the external environment.25. A method as defined in claim 23, wherein: the portable cell growthcassette provided in the element of providing further includes a mediacontainer configured to carry a growth medium, and a waste containerconfigured to carry media discharged from the growth chamber; andconditioning the portable cassette while it is positioned on the secondinstrument platform further includes controlling the flow of growthmedia from the media container through the cell growth chamber to thewaste container, and providing a flow of a gas containing oxygen to thecell growth chamber and a flow of discharge gases from the cell growthchamber.
 26. A method as defined in claim 21, wherein: the portable cellgrowth cassette provided in the element of providing further includes amemory device that carries information relating to the desiredconditioning of the cell growth chamber over time; and conditioning theportable cassette while it is positioned on the first instrumentplatform includes retrieving information from the memory device of theportable cassette and conditioning the cassette based on the retrievedinformation.
 27. A method as defined in claim 21, wherein: the portablecell growth cassette provided in the element of providing furtherincludes an updatable memory device; and conditioning the portablecassette while it is positioned on the first instrument platformincludes storing information to the memory device of the portablecassette, such information relating to the condition of the cassettewhile it is so positioned.